Conventional methods for labeling nucleotides are straightforward, but have significant drawbacks. With direct fluorophore labeling, the bulky dye molecule on the nucleotide makes it difficult for the enzyme to incorporate nucleotides into DNA or RNA strands. Additionally, protocols optimized for one fluorophore may not be optimal for another, chemically different fluorophore.
Various methods have been used to generate labeled probes for hybridization to Southern blots and microarrays, for example, 5′ and 3′ end labeling with 32P. Additionally, nick translation uses DNAse I to generate single stranded nicks in the nucleic acid starting material and DNA polymerase to fill in the nicks. A labeled deoxynucleotide (for example dUTP-digoxigenin or dUTP fluorescein) is included in the reaction mixture, along with the other unlabeled deoxynucleotides. While these methods generate labeled probes, they do not provide a method of amplifying the starting material.
Polymerase chain reaction (PCR) in the presence of a mixture of nucleotides (for example dATP, dCTP, dGTP, dTTP, and modified dUTP-digoxigenin or fluorescein) can be used to synthesize copies of a template strand. These amplicons can be used as probes for hybridization assays. The mixture must contain unmodified dTTP in addition to modified dUTP in order for the reaction to take place.
PCR uses a double stranded DNA template as starting material. This template can be made from RNA by reverse transcription and subsequently labeled by PCR incorporation of labeled nucleotides. While this method does result in an amplification of the starting material, the substitution of the deoxynucleotide fluorescent analogue is less than 100% and the specific activity may be variable, depending on the label.
Accordingly, one object of the present invention is to provide an improved method for nucleotide labeling that circumvents problems associated with conventional methods. Preferably, the methods will be amenable to a variety of uses including generating FISH probes, generating probes for Southern blots, generating probes for Northern blots, calorimetric in situ hybridization probes (CISH), in situ PCR, isothermal amplification in situ, DNA fingerprinting, and SNP detection.